The present invention relates generally to detecting the presence of genetic polymorphisms or mutations associated with Charcot-Marie-Tooth Disease Type 1B (CMT1B). More particularly, the present invention provides compositions and methods for identifying CMT1B-associated polymorphisms or mutations in patient nucleic acid samples by amplification of patient nucleic acid and identifying CMT1B-associated point mutations in the amplification products.
Over 2000 human diseases are known to result from DNA alterations including deletions, multiplications and nucleotide substitutions. Finding genetic disease alterations in individuals and following these alterations in families provides a means to confirm clinical diagnoses and to diagnose disease in carriers, preclinical and subclinical affected individuals, affected unborn fetuses, fetal cells in maternal blood, and preimplantation embryos. Counselling based upon accurate diagnoses allows patients to make informed decisions about potential parenting, ongoing pregnancy, and early intervention in affected individuals.
Disease associated deletions, multiplications, and nucleotide substitutions may be large or small. Polymorphisisms at disease sites can be used to trace abnormal allelic segregation. However, normal polymorphic nucleotide changes can complicate detection of abnormal alleles with changes at different nucleotides. Because multiple alleles within genes are common, one must distinguish disease-related alleles from neutral (non-disease-related) mutations. Most alleles result from neutral mutations that produce indistinguishable, normally active gene products or express normally variable characteristics like eye color. In contrast, some alleles are associated with clinical disease like sickle cell anemia. Disease-related mutations may result from a single point mutation as occurs in sickle cell anemia.
Previously diagnosis and confirmation of genetic disease and carrier states often relied upon enzyme activity testing, statistical analysis, or invasive diagnostic procedures. For example, painful nerve conduction tests have been necessary to detect preclinical and subclinical cases of Type 1 Charcot-Marie-Tooth Disease. Such invasive physiological testing is not available for identifying disease in fetuses.
DNA polymorphisms or mutations (RFLPs; Restriction fragment length polymorphism or mutations) have been used to diagnose more than 20 genetic diseases (See Lebo et al, Am. J. Hum. Genet. 47:583-590, (1990)). The DNA Committee of the Pacific Southwest Regional Genetics Network proposed that a prenatal clinical test must be informative in at least 70% of fetuses and must be at least 95% reliable (Ibid.). The percent informative matings are calculated according to Chakravarti and Buetow, Am. J. Hum. Genet. 37:984-997 (1985), with different formulas for autosomal recessive, autosomal dominant, and X-linked genetic disease. Not all matings are informative because parents may be homozygous for neutral DNA polymorphisms or mutations. The proportion of informative matings depend upon 1) the number of different alleles at each gene locus, 2) the relative frequency of each allele in the population (the most informative have more than one common allele), and 3) whether alleles are distributed randomly throughout the population. Finding enough informative polymorphisms or mutations can be very laborious when few or uncommon polymorphisms or mutations are found at a disease locus (Lebo et al, Am. J. Hum. Genet. 47:583-590 (1990)). Using characterized polymorphisms or mutations may be laborious since several often need to be tested. See, e.g., Lebo et al, Am. J. Med. Genet. 37:187-190, (1990). Even then a proportion of uninformative results in some pedigrees are anticipated.
Charcot-Marie-Tooth disease (CMT, Hereditary Motor and Sensory Neuropathy, HMSN) is the most common generic neuropathy with an incidence of 1/2600 (Skre, Clin. Genet., 6:98-118 (1974)). Genetically heterogeneous CMT subtypes are clinically similar with pes cavus, distal muscle weakness and atrophy, absent or diminished deep tendon reflexes, and mild sensory loss. CMT Type I (CMT1, HMSNI) is a demyelinating peripheral neuropathy with slower nerve conduction velocities while Type II (CMT2, HMSNII) is a non-demyelinating neuronal disorder with near normal nerve conduction velocities (Dyck and Lambert, Arch. Neurol. 18:603-618 (1968) and Dyck and Lambert, Clin. Neurol., 18:619-625 (1968)). The more severe CMT1 tends to be manifested in late childhood or adolescence and progress slowly but inexorably (Bird and Kraft, Clin. Genet., 14: 43-49 (1978); Vanasse and Dubowitz, Muscle and Nerve, 4:26-30 (1981)). CMT has been mapped to chromosome 1 (Bird et al., Am. J. Hum. Genet., 34:388-394 (1982); Guiloff et al., Ann. Hum. Genet., 46:25-27 (1982); Stebbens & Conneally, Am. J. Hum. Genet., 34:195 (1982)), chromosome 17 (Vance et al., Expt. Neurology, 104:186-189 (1989); Lebo et al., Am. J. Hum. Genet., 50:42-45 (1992)), chromosome X (Gal et al., Hum. Genet., 70:38-42 (1985); Beckett et al., J. Neurogenet., 3:225-231 (1986); Fischbeck et al., An. Neurol., 20:527-532 (1986); Ionasescu et al., Am. J. Hum. Genet., 48:1075-1083 (1991)), and another autosomal locus (Chance et al., Am. J. Hum. Genet., 47:915-925 (1990)). Chromosome 17 CMT1A is usually associated with a 1.5 Mb duplication (Lupsky et al., Cell, 66:219-232 (1991); Raeymaekers et al., Neuromuscular Disorders, 1:93-97 (1991); Hoogendijk et al., Human Genet., 88:215-218 (1991)) spanning the peripheral myelin protein gene (PMP22; Patel et al., Nature Genetics, 1:159-165 (1992); Timmerman et al., Nature Genetics, 1:166-170 (1992); Othman et al., Nature Genetica, 1:171-175 (1992); Matsunami et al., Nature Genetics, 1:176-179 (1992)). Mutant PMP22 without duplication also results in clinical CMT1A (Valentija et al., Nature Genetics, 2:288-291 (1992)). Genetic mutations associated with CMT1B have not been published. Therefore, methods of genetic screening and diagnosis have not become available to clinicians and patients suspected of having CMT1B. Because genetic testing has not been available, painful nerve conduction studies have been required to make or confirm the diagnosis of CMT1B. Further, no methods of pre-natal testing have been available.
What is needed in the art is a rapid and reliable method to detect genetic abnormalities associated with CMT1B. The method should be applicable to pre- and post-natal patient samples. Use of such a test could provide a means for pre-natal diagnosis of affected fetuses and provide more accurate and less painful diagnosis of living patients. Quite surprisingly the present invention fulfills these and other related needs.